A chain of discovery closes in on the prime mover of cellular freight.This article first appeared in MBL Science, Winter 1986

F YOU DIDN'T KNOW better you might think you were watching footage of rush hour on Boston's Southeast Expressway, filmed from a helicopter. Two-way highways, rotaries and exits seem to be everywhere, tightly packed with tiny, speeding cars. Most of the vehicles travel in one direction, exiting occasionally onto yet another busy thoroughfare. As is typical with rush hour traffic, the movement is often stop and go. Some more fickle travelers can't seem to decide which way they're heading.
All this channeled commotion takes place on a simple television monitor, but the traffic it reveals is of far greater consequence than any commuter's report on the morning news. The speeding vehicles are a sampling of the millions of tiny organelles - microscopic bubbles and sausages of cellular material - that travel within the giant axon of the Woods Hole squid, Loligo pealeii.

The movement of organelles from one point to another is a fundamental process within all living cells. A cell manufactures certain essential products and objects in its interior that must be distributed throughout the cell to insure its survival. How does the cell accomplish this? What is the driving force behind the movement? Are undesirable materials such as viruses transported in a similar manner?

Until recently, only two "motility systems" were known to account for cellular motion. The system responsible for muscle contraction has long been studied, and scientists know that the interaction of two proteins, myosin and actin, is responsible for this movement. Similarly, it is dynein, a protein very different from myosin, that interacts with tubulin to generate the beating motions of cilia, the short hair-like structures present on the surface of many cells. Scientists now believe they have discovered the molecular basis of a third sort of motion - that traffic of organelles within axons known as fast axonal transport.

Study of the axon (the long extension of a neuron that conducts impulses away from the nerve cell body) actually began a century ago. Santiago Ramon y Cajal's "Neuron Doctrine" established that each cell with its own axon is an independent unit, never forming networks with other axons. Cajal convincingly showed that nerve cells are polarized, suggesting that their signal impulses travel from the nerve cell body with its finger-like projections (dendrites) to the axon. At the MBL and Yale University, Ross G. Harrison took the Doctrine a step further during the early 1900's, by culturing neurons and proving that the axon is an outgrowth of the nerve cell itself, not a separate entity as previously thought. J. Z. Young's visit to the MBL in 1936 culminated in a description of the giant axon of Loligo, an accomplishment that excited neuroscientists world-wide. He described an axon visible without the aid of a microscope, and large enough ( 1000 times wider than that of any vertebrate) to manipulate easily. The squid giant axon soon became an excellent model system for the study of neurons in general.

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Experiments to elucidate further the properties of the axon continued, but it wasn't until 1948, with the description by P. Weiss and H. B. Hiscoe of what would later be termed slow axonal transport, that study of the movement of material through axon began. Their "damming" experiment, which involved blocking the flow within the axon, showed that substances were in fact moving in one direction from the cell body through the axon, a process essential, among other things, to the maintenance of the axon's cytoplasmic matrix or "skeleton." But the question of how the axoplasm and its associated proteins were transported from the cell body to the terminus of the axon remained unanswered. Research progressed slowly, despite the abundance of animals and axons, and the eagerness of people studying them.

IT WAS THE ADVENT OF radioisotopic tracers in the early 1960's that finally made possible detailed analysis of slow transport. Amino acids containing a radioactive tag were injected into the nerve cell body, where they are incorporated into proteins. The movement and numbers of these labeled proteins were then traced and counted using biochemical methods.

Using this technique to study slow transport, Raymond Lasek, of Case Western Reserve University, and others in the mid- 1960's, noticed that slow transport accounted only for a part of the movement within the cell. While slow axonal transport involved the "outbound" movement of certain proteins at a rate of I to 2 millimeters per day, Lasek observed, indirectly, that a faster component was moving organelles both away from the cell body and towards it, covering a similar distance at a much quicker rate - in a matter of hours as opposed to days. Lasek's work with radioisotopes agreed with evidence from another new source: a microscopy technique called Nomarski optics. Using cultured frog neurites, William Burdwood and Robert Allen detected movement of large organelles such as mitochondria in the neurons.

It was apparent that there was a great deal more to the traffic within axons than had ever been expected. But the true extent and nature of the movement awaited another breakthrough, provided by a serendipitous turn of a knob. In the winter of 1981 Bob Allen was teaching a course in Optical Microscopy at the MBL. Hoping to save time by allowing the whole class to view a preparation simultaneously, Allen attached a video camera and a television monitor to his microscope. To his surprise, an adjustment of the contrast to reduce background light revealed structures approximately onetenth the size of the smallest visible under a conventional light microscope. Though an electron microscope is capable of imaging even smaller structures, preparations for the electron beam must be fixed - i.e., killed, dried and sliced beforehand. The one-millionth of an inch organelles revealed by video techniques are very much alive.

Allen soon began using the video microscope system to explore a variety of preparations. An examination of the internal fluid - the axoplasm - of the squid giant axon the following summer clearly revealed that there was movement occurring - lots of it and in both directions.

When the news reached Ray Lasek, who had devoted years to the study of axonal transport and the movement of proteins through the axon, he wasn't exactly elated - at least not at first.

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"I was walking across the courtyard at the MBL," he recalls, "and a friend stopped me and told me that Allen, Janis Metuzals, and Ichiji Tasaki had seen the particles in the axoplasm moving. I was crestfallen. But Allen came up later and said 'I want you to stop by the lab tonight.' "

What Lasek saw that evening was a myriad of "submicroscopic" particles moving quickly and continuously through the axoplasm, primarily in the outbound, or anterograde, direction, along what appeared to be linear elements. Such movement of tiny organelles had never before been seen, despite his and others' radioisotopic and microscopic work.

"I knew the movement was there all along," Lasek says in hindsight, "but it was something I could only visualize in my dreams. Seeing it on the screen was the greatest experience of my life."

The confirmation of particle movement within the axon sent waves of excitement through the community. It also paved the way for an endless supply of questions. How did the particles move? What was the force behind this movement? Was it similar to the actin/myosin or dynein/tubulin systems, or was it something completely different? What were the "linear elements" that the organelles apparently travel along?

The first pressing problem seemed to be the system or preparation itself. Perhaps there was an easier way to look at the axoplasm. Lasek and Scott Brady (then of Case Western Reserve University) immediately began collaborating with Allen. Drawing on Lasek and Brady's previous experience with axoplasm preparations, and employing Allen's new system, the three began an extensive investigation of axoplasm which had been squeezed free from the confines of the axon's protective membrane

Would particle movement still occur after the axoplasm had been isolated? Was movement at all dependent upon that membrane? Brady, Lasek, and Allen found that movement continued after extrusion, and that the addition of "buffer X" which contained ATP - a known "fuel" for the other two motility systems - maintained the structure of the axoplasm for up to 24 hours. Electron micrographs confirmed that the isolated axoplasm was morphologically the same as that found within the axon. Finally they had found a hardy system, the same as that found within a living axon, which could be worked with easily in the laboratory for hours.

BY THE FOLLOWING summer, Lasek and Brady had acquired their own video system and were busy studying isolated, intact axoplasm. Allen focused mainly on dispersed isolated axoplasm, eventually announcing at the 1983 General Meetings of the MBL that when buffer X was added to this axoplasm preparation filaments (the "linear elements") could be seen separately from the rest of the axoplasm. In this dispersed state, the vesicles continued their indefatigable movement in both directions along the isolated filaments. It didn't seem to matter whether the particles and filaments were found within the protective walls of an axon, isolated free from those walls, or separated from one another.

The next question seemed obvious. What were those filaments on which the organelles seemed to be traveling?

Ronald Vale (Stanford University) and Michael Sheetz (University of Connecticut) approached Allen that summer at the MBL. They were interested in testing a hypothesis that the filaments were actually actin bundles, and that movement in this system was analogous to that found in actomyosin (muscle) systems. Allen felt sure that the filaments were microtubules - thread-like fibers containing tubulin - but his experiments had so far been inconclusive. Eventually he agreed to test the actin hypothesis, and the three investigators went ahead with an experiment which used myosin-coated plastic spheres as substitutes for the organelles. If the filaments were actin bundles, the spheres would react with them and move. But the ersatz vesicles simply lay inert.

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Still convinced that the filaments were microtubules, Allen continued to search for an answer. Meanwhile, Vale and Sheetz moved on to the NIH's Laboratory of Neurobiology at the MBL. There they worked with Bruce Schnapp and director Thomas Reese, who were also interested in organelle transport and had collaborated earlier with Lasek and Brady on the freeze fracture of intact axoplasm, a technique used to prepare samples for electron microscopy. Schnapp, Sheetz, Reese and Vale soon began working with the buffer X and axoplasm preparation. As Allen had shown earlier, the addition of buffer X resulted in a limited separation and accumulation of filaments from the axoplasm along the edges of a microscope slide. After trying various combinations of buffers, the group finally found that the addition of water-diluted buffer X to the axoplasm resulted in the dissociation (throughout the slide) of a greater number of filaments which sustain long-lasting organelle movement. With this diluted buffer, the investigators were then better able to visualize and subsequently analyze the filaments.

By the early winter of 1984, Bruce Schnapp and his co-workers and Allen and his colleagues had submitted independent papers to Cell and The Journal of Cell Biology, respectively, confirming that the filaments were microtubules which supported organelle movement in both directions in the presence of ATP. Organelles were seen moving in both directions along individual microtubules, indicating that each microtubule contained more than one track along which numerous organelles might travel. Furthermore, as the investigators had suspected, the microtubules were found to move on their own, crawling along the surface of a small piece of glass when ATP was added to the preparation.THE ROADWAY FOR THE transport of important vehicles and their cargo within the axon had thus been established. The linear tracks were microtubules, and ATP appeared to be the necessary fuel for motion. The identity of the vehicles' engines, however, remained a mystery. Many speculated that the protein dynein was somehow involved in axonal transport since it is associated with microtubules. But results were inconclusive at best. Each advance was leading only to new perplexities.

Back-tracking a bit, the summer of 1984 brought Lasek and Brady together again in a lab at the MBL. Knowing that both organelle and filament movement appeared to be fueled by ATP, they performed a series of experiments using ATP analogues (substances very similar to but not identical with ATP) and the isolated axoplasm preparation. They found that addition of a particular analogue, a substance known as AMP-PNP, would freeze or inhibit organelle movement almost instantaneously. In other words, AMP-PNP partially replaces the ATP in the system, causing the system to come to an abrupt halt. Addition of ATP starts the system running again. Lasek and Brady found that besides replacing ATP, AMP-PNP caused organelles to bind to the filaments, resulting in a "freezing" of the axoplasm. They noted that release from the frozen state could not occur without hydrolysis (cleavage) of the ATP molecule, a property which distinguished this cell movement from that of muscle or ciliary motion. It was beginning to look as though the force behind axonal transport might not be supplied via dynein or myosin after all.

Vale, Sheetz, Schnapp and Reese eventually found a way to separate the axoplasm into three distinct parts: a microtubule fraction, an organelleenriched fraction, and a soluble "supernatant fraction." In tests of varying combinations of the fractions, it soon became apparent that both the crawling of microtubules along a small piece of glass and organelle movement along a microtubule were dependent not only on ATP, but on something found within the soluble supernatant fraction. Vale and his colleagues hypothesized that that "something" was a protein, having observed that the movement was inhibited by heating the supernatant, or by adding the enzyme trypsin - two treatments known to break down proteins. This group had already suspected that a single protein was likely to be responsible for these movements, since they had observed that different sized particles and microtubules all move at the same speed in the same direction. It seemed quite possible that the description of a motor responsible for the transport of material within an axon was within reach. Sifting through the supernatant for just the right "something," however, was a formidable task.

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By early spring Vale and his coworkers had succeeded. Using a biochemical method which filters out unwanted substances, they were able to partially purify, from both cow and squid brains, a protein which appears to induce microtubule and organelle movement. The principal step during the purification process, note Vale, Reese, and Sheetz in the August 1985 issue of Cell, was based on the "frozen axoplasm" results of Lasek and Brady in their experiments with AMP-PNP. It now appears that the newly discovered protein, which they named "kinesin" (from the Greek word meaning "to move"), may attach to organelles being moved along microtubules. The investigators believe that a new cell motility system has been found, one which incorporates a mechanism structurally and enzymatically unlike either of the previously known cellular "motors," dynein and myosin.

YEARS OF INTERACTION and lively scientific exchange of information at the MBL have culminated in this recent and notable discovery. But the identification of kinesin by no means closes the books on the study of organelle transport. (Some investigators, in fact, are not convinced that kinesin is the motor behind axonal transport.) Kinesin supports movement only in one direction: is another protein responsible for the bi-directional movement of organelles? How is movement regulated? How does an organelle know to go to one end of a nerve or the other? Does kinesin exist in other systems?

Scott Brady, now at the University of Texas, has independently reported the partial purification of a similar protein in chick brain. The protein is structurally homologous to kinesin but possesses an important characteristic of motile proteins - the ability to generate energy by cleaving ATP molecules - a property which has not been attributed to kinesin. Another kinesin-like protein has been found in sea urchin eggs by researchers at the University of Colorado; it appears to be involved in the movements of the mitotic spindle during cell division. And in the December 1985 issue of Cell, the Reese group reported that a "crude solubilized fraction" from axoplasm induces the movement of plastic beads along microtubules in the opposite direction from that of kinesin. Their results indicate that there is a second protein which is "pharmacologically and immunologically distinct from kinesin. "

Kinesin molecules (in red) bind to the surface of a spherical organelle and move it along a microtubule.
Meanwhile, investigators such as Anthony Breuer of the Cleveland Clinic Foundation are doing research on axonal transport to learn more about debilitating human neurological diseases such as lateral sclerosis (Lou Gehrig's disease) and Alzheimer's disease. Both may involve abnormal axonal transport processes. The availability of antibodies to kinesin now enables investigators to test for possible defects in kinesininduced transport in each of these diseases.

"Some of the most basic biological processes depend upon transport," notes Bob Allen. "We've stumbled on the solution to lots of mysteries about the function of microtubules in cells including axonal transport and a host of other processes."

MBL investigators will continue to piece together the puzzle that is cell motility. As always in science, one open door simply leads to others, each awaiting the turn of a special key.

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Pamela Clapp was Assistant Editor of the Biological Bulletin at the time this article was published.
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